Silk protein is regarded as a natural biomaterial, and developments of new uses of silk threads in various fields other than that of clothing are underway. Silk fibroin has very good properties such as high strength and flexibility, biocompatibility, blood compatibility, water permeability and oxygen permeability, so it can be used in surgery as implant material and in tissue engineering applications. In addition, silk fibroin can be used as a cell culture matrix, as a substratum for cultivation of cells, as a burn wound dressing membrane, as an enzyme-immobilization material, and in an oral dosage form.
Silk is mainly composed of fibroin and sericin, and the fibroin has 19% hydrophilic side chains containing a heavy chain with a molecular weight of 325 kD (45% glycine, 30% alanine, 12% serine) and a light chain with a molecular weight of 25 kD (15% asparate, 11% glycine, 14% alanine, 11% serine). In addition, the sericin in silk fibers is in an amount of around 25% by weight and it has 76% hydrophilic side chains. The sericin will cause inflammation, so the silk fibers should be degummed with hot water containing surfactants before medical applications. Fibrous products based on silk fibroins have excellent mechanical properties. Nevertheless, silk fibroin has Tg ranging from 170-175° C. and the temperature of transiting random-coil to β-structure conformation is 212° C. Therefore, when the temperature rises to 280° C., the silk fibroin will start to cleave. Thus, silk fibroin is usually processed by solution process rather than by melt process.
To conduct the solution process, silk fibroin need to be dissolved in a salts containing aqueous solution first. U.S. Pat. No. 5,252,285 indicates that fibroin is known to be soluble in certain high ionic strength aqueous salt solutions, for example, aqueous lithium thiocyanate (LiSCN), sodium thiocyanate (NaSCN), calcium thiocyanate (Ca(SCN)2), magnesium thiocyanate (Mg(SCN)2), calcium chloride (CaCl2), lithium chloride (LiCl), lithium bromide (LiBr), zinc chloride (ZnCl2), magnesium chloride (MgCl2), copper salts such as copper nitrate (Cu(NO2)2), copper ethylene diamine (Cu(NH2CH2CH2NH2)2 (OH)2) and Cu(NH3)4(OH)2 and Ajisawa's reagent (CaCl2/ethanol/water). The processing of silk fibroin solution is difficult due to salt concentration increasing when solvent evaporates at an elevated temperature. Even after the salts removed by dialysis out of such aqueous salt/fibroin solutions, the concentration of this fibroin solution is usually too dilute to spin fiber threads. More commonly, the organosoluble silk fibroin is first harvested by freeze drying process from the dialyzed solution. Then, fibers can be spun from fibroin solution dope that was prepared by dissolved silk fibroin solution in an organic solvent. US 2007/0187862 A1 provides for concentrated aqueous silk fibroin solutions and an all-aqueous mode for preparation of concentrated aqueous fibroin solutions by dialysis that avoids the use of organic solvents, direct additives, or harsh chemicals. This application indicates that dialysis of the solution against a hygroscopic polymer is also sufficient to control water content in the formation of silk hydrogels.
Concerning the bottleneck of silk fibroin mass-production, the dialysis process is usually time-consuming and difficult to scale up. Moreover, during the dialysis procedure, the intermolecular hydrogen bonds of silk fibroins gradually form, so molecules of silk fibroins tend to form crystals where the second structure of silk fibroin gradually becomes silk crystal form I (organic solvent soluble) and silk crystal form II (organic solvent insoluble) from random-coil conformation. Furthermore, gelation of silk fibroin renders its second structure unstable, so the solubility of regenerated silk fibroin is hard to maintain.
Sung-Won Ha et al. dissolves silk fibroin with the calcium nitrate tetrahydrate-methanol system and uses wet spinning method to spin regenerated fibroin fiber (Biomacromolecules, 2003, 4 (3), pp 488-496). A solvent system is developed to use a solution containing 1-butyl-3-methylimidazolium chloride as solvent, which can dissolve silk fibroin without dialysis process (Phillips, D. M.; Drummy, L. F.; Conrady, D. G.; Fox, D. M.; Naik, R. R.; Stone, M. O.; Trulove, P. C.; De Long, H. C.; Mantz, R. A. Journal of the American Chemical Society, 2004, 126, 14350-14351). However, this solvent system is expensive and is not appropriate for industrial production. U.S. Pat. No. 7,285,637 applies a formic acid solution containing a small amount of salts to break the disulfide bonds between heavy (350 kDa) and light (27 kDa) chains of silk fibroin so that the dissolution of the silk fibroin in the solution can be facilitated. Nevertheless, there is a serious molecular chain cleavage of silk fibroin in the solvent system. Furthermore, it was reported that silk fibroin can dissolve in hexafluoroisopropanol solvent after 5 months (Zarkoob, S.; Reneker, D. H.; Ertley, D.; R. K. Eby; Hudson, S. D. Synthetically spun silk nanofibers and a process for marking the same, 6110590, 2000). The long dissolving time makes the method impractical for mass production.
Therefore, there is still a need to develop a more convenient and efficient process for producing a silk fibroin.